2. HIV Cellular Pathogenesis I Benhur Lee, M.D.

3. Adults and children estimated to be living with HIV/AIDS as of end 2001 Western Europe 560 000 North Africa & MiddleEast 440 000 North America 940 000 Caribbean 420 000 Sub-Saharan Africa 28.1 million South 6.1 million & South-East Asia 6.1 million Latin America 1.4 million Total: 40 million Eastern Europe & Central Asia 1 million Australia & New Zealand 15 000 East Asia & Pacific 1 million

4. Estimated number of adults and children newly infected with HIV during 2001 Eastern Europe & Central Asia 250 000 East Asia & Pacific 270 000 Australia & New Zealand500 Sub-Saharan Africa 3.4 million South & South-East Asia 800 000 Western Europe 30 000 North Africa & Middle East 80 000 North America 45 000 Caribbean 60 000 Latin America 130 000 Total: 5 million

5. Estimated adult and child deaths from HIV/AIDS during 2001 Sub-Saharan Africa 2.3 million East Asia & Pacific 35 000 Western Europe 6 800 North Africa & Middle East 30 000 Eastern Europe & Central Asia 23 000 South & South-East Asia 400 000 Australia & New Zealand120 North America 20 000 Caribbean 30 000 Latin America 80 000 Total: 3 million

6. See Quicktime Movie file

7. HIV: 15 proteins (9 genes) and an RNA ~10 kb Pox viruses: >100 ORFs ~200 kb (e.g. Smallpox)

8. Lipid Membrane Derived From Host Cell

11. Reverse Transcription &

12. Productive uncoating requires a cellular chaperone protein (Cyclophilin A) Chaperones –facilitate protein folding and assembly –has peptidylprolyl isomerase activity Viruses made without cyclophilin A cannot undergo reverse transcription but are fusion competent –Point mutations in capsid that cannot bind cyclophilin A –Viruses produced in the presence of Cyclosporine A Efficient RT may require expansion or partial disassembly of the viral capsid core

13. RT (-)Strong-stop DNA (+)Strong-stop DNA

15. Pre-Integration Complex

18. Integration 1)Processing (Integrase) 2)Joining (Integrase) 3)Repair (Host cell)

19. RU5 Reverse Transcription Products: Early Late Cytoplasm Nucleus

20. LTR Transcription (tat)

22. rev-mediated

23.  Requires protease (PR) to process structural proteins (Gag)  Protease Inhibitors prevent viral assembly (used in combination with RT-Inhibitors in HAART)  Requires human/primate co-factors: Gag processing is deficient in murine cells  Assembly and budding takes place at lipid rafts: cholesterol-rich membrane microdomains

26.  CD4 is necessary but not sufficient for viral entry  CD4 mediates viral attachment to cell surface but not membrane fusion

27. Coreceptor A Coreceptor B Coreceptor A Coreceptor B (Early) (Late)

28. CD8+ T-cells CD4+ T-cells Supernatant HIV-1 No Viral Replication ? CAF (Cellular Antiviral Factor)

29. CD8+ T-cells RANTES (CCL5) MIP-1  (CCL3) MIP-1  (CCL4) Chemokines HPLC Fractionation Viral Inhibition Activity Fraction # 1 45 Purify, Mass Spec or Protein sequencing Database searching Biochemical Confirmation WB, IP, and Neutralization Expt. Supernatant

30. Chemokines --chemotactic cytokines --8-10 kDa --binds to 7-TM GPCR --classified based on number and spacing of conserved cysteine residues --CC, CXC, CX 3 C, XC

32. CAF (Cellular Antiviral Factor)

33. Expression Cloning Identifies a Chemokine Receptor as Coreceptor for T-tropic Viral Entry

34. Effector Cell Transfect pools of cDNA library Target Cell CD4 Cell-Cell Fusion vacEnv vacT7-Pol vacT7-  -gal T7  -gal HIV Env (gp160) T7  -gal T7-Pol

35. Expression Cloning Identifies a Chemokine Receptor as Coreceptor for T-tropic Viral Entry 1000 cDNA pools (@ 4x10 3 each) 1000 cDNA pools (@ 4 each) Screen Plasmids from Individual Colonies Sequence positive plasmid

36. Confirmation of Coreceptor Function of Fusin (CXCR4) Fusion induced only when expressed with CD4 Fusion not induced with uncleaved (non- functional) Env Fusin (CXCR4) expression confers cell fusion- promoting activities in a variety of CD4- expressing cell lines (that were otherwise non- permissive) Endogenous expression of Fusin in a variety of permissive cell lines Antibody to Fusin inhibits cell fusion

37. CCR5 binds RANTES, MIP-1  & MIP-1 

38. Use of Different Coreceptors Largely Accounts for Viral Tropism

39. ccr5/ccr5 81% of Caucasian Get infected normally Progress to AIDS normally ccr5/  ccr5 15-18% of Caucasians Get infected normally But progress to AIDS 2-4 years more slowly  ccr5 /  ccr5 1% of Caucasians Highly Resistant to Infection Loss of CCR5 function but otherwise normal (no side effects) This makes CCR5 an attractive drug target

40. CXCR4 SDF-1 CXCR4/SDF-1 Knock-Out Mouse phenotype is lethal Chemokine Ligand SDF-1[3’A/3’A] SDF-1[+/3’A] SDF-1[+/+] Polymorphism in 3’ untranslated region of SDF-1 gene may be associated with delayed progression to AIDS

41. CCR5 and CXCR4 are the major Coreceptors for HIV-1/HIV-2 entry All HIV-1/HIV-2/SIV strains use either CCR5, CXCR4 or both SIV can efficiently use alternate coreceptors: GPR15(BOB), CXCR6(Bonzo/STRL33), GPR1 Polymorphisms in chemokines or their receptors have been reported to modulate the course of HIV disease. Other Chemokine receptors with Coreceptor activity (in vitro) –CCR2 –CCR3 –CCR8 –CX3CR1 –APJ –US28 (from CMV)

42. Experimental Strategies for Identification of Viral Cellular Receptors

43. Impact of Coreceptor Discoveries On: Virus Entry Mechanism: structure-function, how does Env interact with coreceptor, can fusion be inhibited? (Lee et al., Science 281:487; Lee et al., J. Biol. Chem. 274:9617; Blanpain and Lee et al, J. Biol. Chem., 274: 18902; Wang and Lee et al., J. Biol. Chem. 274: 28413, Blanpain et al., Blood, 94: 1899) Pathogenesis:does differential utilization (expression) of coreceptors account for the variable outcomes and pathological syndromes associated with HIV infection ( Lee et al, Blood 93: 1145; Hariharan et al, Blood 93: 1145; Lee et al., Proc Natl Acad Sci USA 96: 5215; Honczarenko et al., Blood 94: 2990, Majka et al., J. Clin. Invest. 104: 1739, Lee et al., Eur.J. Immunol., in press) Coreceptor Polymorphisms: consequences for disease progression (CCR2-V64I, CCR5 promoter) (Lee at al., J. Virol.72: 7450; Martin et al., Science 282: 907)

45. >90% >75% >50% Mutations affecting gp120 Binding to CCR5 Degree of Conservation Most Least CD4 gp120 Binding

46. Virus Membrane Cell Membrane

47. Small Molecular Antagonists 1)TAK779 2)Sch-C 3)E914 Modified Chemokines 1)AOP-RANTES Monoclonal Abs

48. Entry of HIV is triggered by receptor engagement

49. Viral Attachment Factors

50. DC-SIGN (CD209) C-type (calcium-dependent) lectin Relatively specific expression on subsets of dendritic cells in tissue Binds to HIV Env (gp120) with greater affinity than CD4 (Kd 1.3nM vs 4.5 nM) Binding to gp120 can be competed off with certain sugars (mannan>L-fucose>  - methyl-D-mannoside)

51. DC-SIGN: A Conduit for Transfer of HIV to Lymphoid Organs? Geijtenbeek, T.B.H. et al, Cell 100: 594

52. DC-SIGN mediates HIV-1 infection in DC-T-cell co-culture DC-SIGN enhances HIV infection in trans

53. Consequences of DC-SIGN Mediated High Affinity Viral Attachment Altered or expanded viral tropism due to infection of target cells with –Lower CD4 levels –Lower coreceptor levels –Alternate coreceptors (e.g. Bonzo/STRL33)

54. Some DC-SIGN transduced T-cell lines can support R5-Viral Replication JurkatSupT1 Jurkat and SupT1 cells do not express detectable levels of CCR5 by FACS

55. R5 Viral Infection of Jurkat/DC-SIGN+ cells are CD4 and CCR5 dependent (CCR5 Antagonist)